The cell wall of many species of Lactobacillales consists of multiple peptidoglycan layers decorated with serotype-specific polysaccharides that are characterized by the presence of rhamnose. Streptococcus mutans is a key etiological agent of human dental caries, and has also been implicated in bacteremia and infective endocarditis. Based on the chemical structures of serotype-specific carbohydrates, S. mutans is classified into serotypes c, e, f and k with approximately 70-80% of strains found in the oral cavity classified as serotype c. The serotype c-specific carbohydrate (SCC) of S. mutans is composed of a polyrhamnose backbone with ?- linked glucose side-chains. Although carbohydrate structures have been reported for all S. mutans serotypes, our recent insights into the structure of these glycopolymers indicate that the functionally important modification, glycerol phosphate, was overlooked, possibly due to its loss during the purification steps. This new finding justifies a re-examination of the chemical structures of serotype-specific carbohydrates. One of the goals of the proposed study is to determine the molecular structure of SCC isolated from S. mutans using mild, non-destructive methods. Moreover, little is known about the multienzyme processes involved in attachment of the glucose side-chains to the polyrhamnose backbone of SCC and the function of the glycerol phosphate modification in S. mutans. Our preliminary findings revealed that the glycerol phosphate modification plays important roles in S. mutans morphology, autolysis, resistance to antimicrobials and biofilm formation. We propose to identify and functionally characterize the enzymes involved in glucose side-chain attachment to polyrhamnose, and investigate the molecular mechanisms underlying the functions of glycerol phosphate modification in morphology, autolysis and biofilm formation. To accomplish our goals, we will employ streptococcal genetics, in vitro enzymology, NMR spectroscopy of polysaccharides, analytical chemistry, mass spectrometric analysis of phospholipids, and various methods of microscopy including AFM-based nanomechanics. The cell wall biosynthetic machinery is an historically preferred target for the development of novel antimicrobials. In order to validate the pathway of SCC decoration with glycerol phosphate as a potential drug target, we will determine the role of this modification in a rat caries model. Successful outcomes will guide future studies of cell wall biogenesis in other important Gram-positive bacteria and the development of novel strategies to treat S. mutans infections.